Many factors influence the quality of citrus products, including juices. To consumers, taste, flavor, acidity, bitterness or tartness, color, and texture are important considerations for acceptance of these products. Indeed, consumer acceptance is a critical factor to the citrus industry. This is of particular importance in view of the world market for citrus products. For example, the world production of orange juice has reached an excess of 900 million single strength gallons (see, S. I. Norman and D. A. Kimball, Trans. Citrus Engineer. Conf., ASME 36:1-31 [1990]), and for grapefruit juice, the annual consumption is approximately 100 million single strength gallons (see, S. I. Norman and D. A. Kimball, Trans. Citrus Engineer. Conf., ASME 36:1-31 [1990]). As discussed below, the principal bitterness compounds in grapefruit and orange juices are naringin and limonin, respectively. The predominant acid which is perceived as the tartness of the citrus juice is citric acid (Charles et al., Food Sci., 51:415 [1986]). Sensory properties of the sour orange (Citrus aurantium) juice are extremely different from those of sweet orange juice (Couture and Rouseff, J. Food Sc., 57:380 [1992]). The sourness of sour oranges is due to an elevated acidity, especially in comparison with sweet oranges. It has been reported that excessive acidity reduces the consumer acceptance of grapefruit juice (See, Johnson and Chandler, Food Technol., May, 1988, pp. 130-137). Thus, sour orange juice and grapefruit juice are viewed as less commercially important than sweet juice.
Bitterness in citrus juice products (e.g., orange and grapefruit juices) has been a long-standing consumer acceptance problem in the industry. The presence of bitterness in citrus fruit juices has resulted in attempts to limit or avoid the development of bitterness in the fruit used to produce juice. For example, because of the development of bitter taste that occurs in early to mid-season oranges and early season grapefruits, these fruits are rarely used in the production of juice. Thus, the presence of bitterness represents a significant loss in the amount of fruit available and suitable for use by the juice industry. In order to permit the use of early to midseason fruits, as well as fruit varieties that naturally are more bitter than others, much research has been conducted to identify the chemical compounds responsible for the bitterness of citrus juices. This research has identified two major classes of compounds responsible for the bitterness of citrus juices, namely the limonoids and flavonoids.
Liminoids
The limonoids comprise a class of chemical compounds widely distributed in all citrus species (V. P. Maier et al, in Citrus Science and Technology, S. Nagy et al. (eds.), AVI Publishing Co., Westport, Conn., 1:355-396 [1977]; and V. P. Maier et al., Citrus Nutr. Qual., 143:63-82 [1980]). The most important limonoid compound, limonin, is an intensely bitter compound that is of commercial significance in the citrus industry, as even low concentrations of this compound (e.g., 6 parts per million) may cause significant reductions in juice quality.
Limonin is a highly oxygenated triterpene dilactone, with the chemical formula C.sub.26 H.sub.30 O.sub.8, and a molecular weight of approximately 470, and a volume of approximately 402 cubic angstroms. As shown in FIG. 1, limonin includes an epoxide, two lactone rings, a five-membered ether ring, and a furan ring. All other citrus limonoids have been reported to have the furan ring and at least one of the lactone rings (V. P. Maier et al., supra). Limonin is only slightly soluble in water and alcohol, although its water solubility is increased in the presence of sugar and pectin. It is soluble in glacial acetic acid, acetonitrile, and chloroform.
Limonin is produced as an esterification product of limonoic acid A-ring lactone. During extraction of citrus juice from fruit, this non-bitter lactone compound undergoes an enzyme-induced, acid-catalyzed, esterification to form the bitter compound known as limonin. During pasteurization and/or evaporation of the juice, heat catalyzes the esterification reaction. This production of limonin from limonoic acid A-ring lactone is often referred to as "delayed bitterness."
In addition to limonin, another limonoid known as "nomilin" has also been associated with delayed bitterness (see e.g., R. L. Rouseff, J. Agricult. Food Chem., 30:504-507 [1982]). However, it is usually present in much lower concentrations (e.g., &lt;2 ppm) in citrus juice. Thus, it is of less commercial significance than limonin (S. I. Norman and D. A. Kimball, Trans. Citrus Engineer. Conf., ASME 36:1-31 [1990]).
Flavonoids
Flavonoids are chemicals that, like the limonoids, are widely distributed throughout the higher plant kingdom. There are two flavonoids (hesperidin and naringin) of particular importance as quality indicators for the citrus industry. For example, if it is present in citrus juice, hesperidin precipitate results in lower juice quality (see e.g., Norman and Kimball, supra). In addition, high concentrations of naringin also reduce juice quality (see e.g., Norman and Kimball, supra). The highest concentrations of naringin are found in the albedo portion of the citrus fruit, while the highest concentrations of limonin are found in the seeds and rag.
Naringin, with a molecular weight of approximately 580, an approximate volume of 465 cubic angstroms, and chemical formula C.sub.27 H.sub.32 O.sub.14, is composed of one flavonoid group attached to a disaccharide (glucose-rhamnose). The structure of naringin is shown in FIG. 2. If the rhamnose is attached at the C-7 position of the flavonoid, the compound is bitter. However, if the rhamnose is attached to the C-2 position of the flavonoid, the compound is tasteless (R. L. Rouseff, in "Citrus Nutrition and Quality (S. Nagy and J. A. Attaway, eds.), ACS Symposium Series 143:63-65 [1980]). Thus, much research has been conducted regarding the various forms and isomers of the flavanones (see e.g., R. L. Rouseff, supra). While naringin is only slightly soluble in water, it is soluble in acetone, alcohol, and warm acetic acid.
Debittering of Juice
Both pre-harvest and processing methods have been investigated for reducing the bitterness of citrus fruit, as well as methods for debittering harvested juice, in order to improve the flavor and enhance the commercial value of the juice. The use of plant growth regulators, rootstocks and other horticultural factors (see e.g., R. F. Albach et al., J. Agric. Food Chem., 29:313-315 [1981], as well as post-harvest fruit treatment with ethylene (V. P. Maier et al., Citrograph 56:373-375 [1971], and the use of low pressures during juice extraction to prevent albedo disruption (J. H. Tatum and R. E. Berry, J. Food Sci., 38:1244-1246 [1973] have been studied as methods to control bitterness.
Adsorbents and ion-exchange resins have also been used. For example, activated carbon has been used to debitter orange juice. (See e.g., R. J. McColloch, Calif. Citrograph 35:290-292 [1950]; and U.S. Pat. No. 2,510,797). Polyamides have also been used (see e.g., B. V. Chandler et al., J. Food Agricult., 19:83-86 [1968]; and M. O. Nisperos and G. L. Robertson, Philip Agriculture 65:275-282 [1982]). Other adsorptive agents have also been used with varying success to debitter citrus juices, including cellulose acetate (B. V. Chandler and R. L. Johnson, J. Sci. Food Agricult., 28:875-884 [1977], cellulose esters (B. V. Chandler and R. L. Johnson, U.S. Pat. No. 3,989,854); Florisil (C. R. Barmore et al., J. Food Sci., 512:415-416 [1986], cyclodextrin polymers (P. E. Shaw and B. S. Buslig, J. Agricult. Food Chem., 34:837-840 [1986], ion exchange resins (see e.g., Kunin U.S. Pat. No. 2,681,907; Gage et al., Science 113:522-523 [1951]; and R. Couture and R. Rouseff J. Food Sci., 57:380-384 [1992], including polyhexamethylene adipamide and polyvinylpyrrolidone (U.S. Pat. No. 3,463,763), and styrene divinyl-benzene (SDVB) cross-linked copolymer resin (e.g., Mitchell and Pearce, U.S. Pat. No. 4,439,458).
Other methods to debitter citrus juices have included the use of ultrafiltration and adsorption (E. Hernandez et al., J. Food Sci., 57:664-670 [1992]; and M. Wethern, Trans. Citrus Engineer. Conf., ASME 37:48-66 [1991], supercritical carbon dioxide (D. A. Kimball, J. Food Sci., 52:481-482 [1987]), immobilized enzymes (D. Dinelli and F. Morisi, French Patent No. 2,125,539; M. On et al., J. Ferment. Technol., 55:493-500 [1977]; A. C. Olson et al., J. Food Sci., 44:1538-1361 [1979]; and M. C. Gray and A. C. Olson, J. Agricult. Food Chem., 29:1298-1302 [1981], immobilized microorganisms (S. Hasegawa, Food Biotechnol., 1:249-261 [1987], and the use of bitterness modulators (e.g., neodiosmin) (D. G. Guadagni et al., U.S. Pat. No. 4,154,862 [1977]). The use of cellulose esters has been successful in the partial removal of flavonoids (e.g., K. S. Kealey and J. E. Kinsella, in "Critical Reviews in Food Science & Nutrition," 11:1-40 [1979]).
However, despite the large number of methods studied to remove bitterness from citrus juices, all of the previously reported methods have serious limitations. For example, carbon adsorbents are non-specific and consequently remove other components present in the juice (R. E. Berry, Proc. Intr. Soc. Citriculture, pp. 896-899 [1981]). The use of polyamides has a major drawback in that it results in the substantial loss of ascorbic acid from orange juice. In addition, the use of polyamides requires a two-stage treatment of the juice, due to the preferential adsorption of phenolic compounds by polyamines. Thus, this method is not economically viable. The methods involving the use of immobilized enzymes have been hampered by the unavailability of commercial quantities of purified enzymes, low reaction rates associated with immobilized enzymes, and the inadequate half lives of the immobilized enzymes. Methods using immobilized microorganisms also suffer from practical problems.
Thus, there remains a need in the art for debittering and deacidification methods that are economical, efficient, and utilizes materials approved by governmental agencies for food processing (e.g., the Food and Drug Administration [FDA]).